All eukaryotic cells including yeast have an intracellular organelle called the endoplasmic reticulum (ER) which is responsible for the transport of secretory proteins and membrane proteins. That is, secretory proteins to be transported to the outside of the cell not to cytoplasm are translated in ribosomes, and immediately translocated into the ER. In this secretory pathway, the misfolded proteins are accumulated to generate ER stress, which leads to activate a stress response mechanism, UPR, in order to remove the generated ER stress (or secretion stress). UPR has been found in all eukaryotic cells, from a unicellular eukaryotic microorganism, yeast, to the highest eukaryotic organism, human.
The study at the genome level on the UPR mechanism of yeast was carried out with the traditional yeast Saccharomyces cerevisiae (S. cerevisiae) in the 2000s (Cell 101, 249, 2000), which resulted in the identification of underlying regulatory and action mechanisms of UPR (Cell 107, 103, 2001; Nature 415, 92, 2002).
The UPR in yeast was first shown to be mainly regulated by a regulatory transcription factor, Hac1p, which is encoded by the HAC1 gene in S. cerevisiae. However, subsequently, studies on the UPR mechanism in higher organisms have been more actively conducted, leading to the discovery of a variety of regulatory factor such as ATF6 and PERK proteins, in addition to XBP1 protein corresponding to the mammalian homolog of Hac1p.
Yeast, as a unicellular eukaryotic microorganism, has the same intracellular organelles as do higher eukaryotic organisms, and their secretion mechanisms are quite similar each other. Therefore, yeast has been considered as an excellent recombinant protein expression host, in which proteins derived from human can be easily expressed at low cost.
In an animal cell expression system, there is an advantage of expressing complex glycoproteins derived from human. However, the main drawbacks to the system include low productivity, high cost, and a long period of time to develop its cell lines. Moreover, a risk of virus and prion contamination is problematic, since animal cells are very similar to human cells. In contrast, yeast can be a more practical expression system, in that it can easily produce the desired recombinant proteins at lower cost and it has been exploited by Mankind for thousands of years in fermentation process. Currently, several yeast strains possess GRAS (generally recognized as safe) status.
Among various yeast species, the traditional yeast S. cerevisiae has been a subject of biological study, and developed as a host for the production of recombinant proteins. However, S. cerevisiae has lower capacity for the secretory production of functionally active proteins for industrial use, as compared to other industrial yeasts such as Pichia pastoris or H. polymorpha. Accordingly, instead of S. cerevisiae, these non-Saccharomyces yeasts become increasingly used as a host for secretory expression.
The methylotrophic yeast H. polymorpha has recently attracted a great deal of interest as a host having excellent ability in secretory expression of recombinant proteins. A great deal of success using H. polymorpha expression systems has been reported in the production of phytase (13.5 g/L), and hepatitis B vaccine for medical use. In particular, the mass-production of hepatitis B vaccine was achieved due to the economical advantage of H. polymorpha as a production host, so that the recombinant vaccines at cheap price could be supplied to many third world countries, contributing to the improvement in the welfare of all mankind.
H. polymorpha has been internationally recognized as an excellent host system for mass-production of recombinant proteins for industrial and medical applications, and thus the development of recombinant protein production technologies using H. polymorpha is highly expected to be very useful in the field of biotechnology. Accordingly, in industry and market, there is a need to develop H. polymorpha as a host system that efficiently produces high quality of secretory proteins, including glycoproteins derived from human, by improving its ability of expressing and secreting recombinant proteins.
The present inventors have performed basic and applied researches to develop H. polymorpha as a host for secretory expression of recombinant proteins for the past ten years, and recently developed a whole-genome microarray system to analyze the regulatory mechanisms of H. polymorpha at a genome level. In an effort to up-grade a cell remodeling technique for optimizing the ability of secretory expression in H. polymorpha, we have isolated a H. polymorpha HAC1 gene (HpHAC1), which encodes an important regulatory transcription factor involved in UPR, and developed the deletion and overexpression strains thereof, so as to analyze their characteristics. Further, the inventors have performed a microarray analysis for them, so as to provide a basis for understanding a UPR mechanism that relieves the secretion stress, and a method for optimizing the ability of secretory expression in H. polymorpha, thereby completing the present invention.